2D Particle Alignment and Classification

It is usually difficult to discern fine structural details of individual particles in raw images taken on an electron microscope. This is due to the low signal to noise ratio typical for imaging of radiation-sensitive biological samples, and it becomes more challenging when the particles are conformationally or compositionally heterogeneous. However, individual particles can be selected from the images, aligned relative to each other, classified into similar classes and averaged to enhance the signal. The resulting 2D class averages provide a wealth of information on basic particle features, such as their size and shape, domain structure and symmetry. They can also help in understanding their degree of particle flexibility. 2D particle alignment and classification is a convenient and routine method to obtain initial structural information for a given particle prior to embarking on the more demanding procedures required for 3D structure determination.

These analyses help answer questions like:

Size
What is the size range of particles in the sample?

Morphological Analysis
Are particles pleomorphic or is their shape well defined?

Aggregation Assessment
What is the integrity of particles in the formulation? Are solubility issues a result of submicron aggregation?